In retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene (RAX) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated. For this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293 T cells and then in hRPCS derived from human embryonic stem cells. The candidate region (RAX-3258 bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for in vitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells. Furthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

中文翻译：

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含有推定人 RAX 启动子的 EGFP 报告质粒的构建和表征，用于体外监测视网膜祖细胞身份

在视网膜退行性疾病中，进行性和衰弱的病症会导致视网膜细胞恶化和视力丧失。在人类中，视网膜缺乏固有的再生能力。因此，从人视网膜祖细胞（hRPC）再生视网膜层是一项具有挑战性的任务，并且hRPC的体外维持受限仍然是主要障碍。视网膜和前神经褶同源盒基因 (RAX) 在视网膜发育和 hRPC 的维持中发挥着关键作用。在这项研究中，首次对具有潜在启动子活性的人类RAX基因的调控区域进行了实验研究。为此，在对人 RAX 基因调控区进行计算机分析后，首先在 293 T 细胞中评估由推定启动子序列衍生的 EGFP 报告基因的表达，然后在源自人胚胎干细胞的 hRPCS 中评估。候选区域 (RAX-3258 bp) 在 hRPC 中显示出最高的 EGFP 表达。该报告构建体可用于体外监测 hRPC 身份并验证用于维持这些细胞的有效培养基。此外，我们的研究结果为更好地了解人类 RAX 基因的调控区域及其在视网膜发育中重要功能的分子机制提供了一个平台。